畜牧兽医学报 ›› 2011, Vol. 42 ›› Issue (11): 1649-1653.doi:

• 研究简报 • 上一篇    

猪瘟病毒贵州流行株E2基因原核表达及其免疫原性的研究

汤德元1* ,李春燕1,罗险峰2,黄涛1,曾智勇1,甘振磊1,王凤1   

  1. 1. 贵州大学动物科学学院,贵阳 550025; 2. 贵州省动物疫病预防控制中心,贵阳 550008
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2011-11-25 发布日期:2011-11-25
  • 通讯作者: 汤德元

Prokaryotic Expression and Immunogenicity of E2 Gene of CSFV Guizhou Epidemic Strain

TANG Deyuan1*, LI Chunyan1, LUO Xianfeng2, HUANG Tao1,
ZENG Zhiyong1, GAN Zhenlei1, WANG Feng1
  

  1. 1. Department of Animal Science, Guizhou University, Guiyang 550025, China; 2. Animal Disease Prevention and Control Center in Guizhou Province, guiyang 550008, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2011-11-25 Published:2011-11-25
  • Contact: TANG Deyuan

摘要: 本研究根据GenBank登录的猪瘟病毒Shimen和C株的E2基因序列设计1对特异引物,采集来自贵州的疑似猪瘟病死猪组织,提取总RNA,进行RTPCR扩增,将扩增产物测序后进行核苷酸序列比较分析。并将RTPCR产物克隆到pMD18T载体上,构建重组质粒pMD18TE2,经双酶切鉴定、核苷酸序列分析后,将E2基因亚克隆到pET32a(+)原核表达载体上,成功构建猪瘟病毒E2基因原核表达质粒pET32aE2,将构建好的原核表达质粒pET32aE2转化至宿主菌BL21中,诱导表达目的蛋白,表达产物经SDSPAGE电泳、Western blot分析,得到了约58 ku的条带,与预期大小相符,进一步提取、纯化目的蛋白,将目的蛋白加入弗氏佐剂免疫小鼠,免疫后采血检测抗体,结果显示目的蛋白能诱导小鼠产生一定抗体水平,该研究为猪瘟亚单位疫苗的研究奠定了基础。

Abstract: According to published cDNA sequence of E2 gene in GenBank of CSFV Shimen and C strain, the synthesized specific primers was designed. E2 gene fragments of classical swine fever virus Guizhou strain were amplified by extraction tissue RNA of the suspected CSF dead and RTPCR. The PCR products were sequenced and comparatively analyzed. The products of RTPCR were cloned into pMD18T vector to form recombinants pMD18TE2 and subcloned into prokaryotic expressing vector pET32a(+). The pET32aE2 was transformed into the E. coli strain BL21(DE3) and the expression of the E2 protein was induced. After analysis by SDSPAGE electrophoresis of the induced expressed protein and Western blot, we get about 58 kDa protein in line with the expected size. Through further extracting and purifying the target protein, the expressed protein was used to immunize mice by adding adjuvant, and then the antibody titers of mice were detected. The results showed that the target protein induced antiCSF antibody in mice. This study laid the foundation for the research of subunit vaccine of swine fever.